Human epidermal growth receptor polymorphisms (HER1–rs11543848 and HER2–rs1136201) exhibited significant association with breast cancer risk in Pashtun population of Khyber Pakhtunkhwa, Pakistan

Abstract Background and Aims Breast cancer is the most common type of cancer in women. The genetic polymorphism in HER (HER1–rs11543848 and HER2–rs1136201) were found to be associated with breast cancer risk in different ethnicities worldwide with inconsistent results. The aim of this research study was to evaluate the association of HER1–rs11543848 and HER2–rs1136201 polymorphisms as a risk of breast cancer in Pashtun population of Khyber Pakhtunkhwa, Pakistan. Methods A total of 314 women including 164 breast cancer patients and 150 age and gender‐matched healthy controls were enrolled from June 2021 to May 2022. All the samples were subjected to DNA extraction followed by Tetra‐ARMS‐PCR for genotyping and gel electrophoresis. Results Our results indicated that HER1–rs11543848 risk allele A (p = 0.0001) and heterozygous genotype GA (p = 0.0001) displayed highly significant association with breast cancer, while the homozygous mutant genotype AA indicated association but nonsignificant results (odds ratio [OR] = 2.637, 95% confidence interval [CI] = 1.2258–5.6756, p = 0.0833). Similarly, the HER2–rs1136201 risk allele G (p = 0.0023), the heterozygous genotype AG (p = 0.0530) and homozygous mutant genotype GG showed significant association (OR = 2.5946, 95% CI = 0.9876–6.8165, p = 0.0530) with breast cancer risk. Both the SNPs presented a higher but nonsignificant risk of breast cancer in postmenopausal women (OR = 2.242, p = 0.08 and OR = 2.009, p = 0.06). However, both the SNPs showed significant association (p < 0.005) with family history, metastasis, stage, luminal B, and TNBC. Conclusion In conclusion, HER1–rs11543848 and HER2–rs1136201 polymorphisms are significantly associated with the higher risk of breast cancer in Pashtun population of Khyber Pakhtunkhwa, Pakistan. These findings advocate for further exploration with larger datasets, offering promising avenues for personalized approaches in breast cancer research and potentially enhancing clinical practices for better risk assessment and targeted management strategies.

exploration with larger datasets, offering promising avenues for personalized approaches in breast cancer research and potentially enhancing clinical practices for better risk assessment and targeted management strategies.

K E Y W O R D S
breast cancer, HER1&2, genetic polymorphism, genetic predisposition, risk association

| INTRODUCTION
Breast cancer is the most common type of cancer and the leading cause of death in women worldwide. 1Among all the Asian countries, Pakistan has the highest incidence rate of breast cancer, usually one in every nine women has a lifetime risk of being diagnosed with breast cancer. 2There are several causes of breast cancer including lifestyle (diet, obesity, high alcohol, and lack of physical activity), ecological factors (exposure to radiations, and so on), and hereditary factors (genetics and family history) are the most significant risk factors. 3e growth factor receptors are involved in proliferation, differentiation, and survival of many tumor cells. 4A family of epidermal growth factor receptor (EGFR) containing tyrosine kinase domain plays an important role in cell growth, differentiation, and tumorigenesis. 5It consist of four structurally similar tyrosine kinase receptors; HER1 (erbB1 or EGFR), HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). 6The activation of these receptors regulates downstream pathways that help in cellular proliferation, cell differentiation, growth, and survival. 7R1 also known as ErbB-1 or EGFR is a transmembrane protein that causes the activation of downstream pathways resulting in cell division, growth, inhibition of apoptosis, and increasing angiogenesis after stimulation of the HER1 receptor.8 The extracellular domain of HER1 gene consists of 1-14 Exons, the transmembrane region has Exon 15, while the intracellular domain possess 16-20 Exons.9 A ligand known as transforming growth factor alpha (TGF alpha) binds to the extracellular domain of HER1 causing the auto phosphorylation of tyrosine kinase residue which is present in the intracellular region, due to this a downstream cascade is initiated.7 In HER1 single nucleotide variation occurs at 497 codons in which the substitution of Arg (R) to Lys (K) occurs in the extracellular domain and is called R497K polymorphism (rs11543848).However, this mutation is detected in breast cancer as well as gliomas, colorectal and lung cancer.10,11 HER2 is a proto-oncogene also known as neu-oncogene which consists of 1255 amino acids and transmembrane glycoprotein (185 KD) that is located on the long arm of the chromosome 17 (17q12).HER2 or ErbB2 is a member of EGFR that regulates the growth and differentiation of cells through several downstream pathways.12 The HER2 receptor always exists in the form of monomer but when a ligand (HER3, HER4, and HER) binds to its extracellular domain it becomes dimerized causing the transphosphorylation of its intracellular tyrosine kinase domain.Various downstream signaling pathways which are mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate3-kinase (P13K), and protein kinase C (PKC) are activated by HER2 that helps in the regulation of cell growth and proliferation.13,14 HER2-positive breast cancer is highly aggressive and almost 20%-30% of breast cancer is due to the polymorphisms in HER2 gene. 15The overexpression of HER2 initiates an oncogenic signaling cascade by the activation of cytoplasmic tyrosine kinase activity. 16The polymorphisms in HER2 enhance the tyrosine kinase activity as well as increasing the formation of HER2 heterodimers that cause the progression of tumor. 17The polymorphism (I655V) present in the transmembrane region of HER2 is linked with an increased risk of breast cancer. 18At codon 655, the conversion of Ile:ATC to V:GTC causes an increase in dimerization, tyrosine kinase activity, and autophosphorylation leading to cell transformation. 19In HER2-positive Breast cancer nearly 50 copies of HER2 gene causes 40-100-folds rise in HER2 proteins and approximately 2 millions of receptors of HER2 are expressed on the surface of tumor cells. 20In contrast, trastuzumab is a monoclonal antibody that is used for the targeted therapy of HER2-positive breast cancer improving overall survival. 21The correlation of Ile655Val (rs1136201) mutation in HER2 with the higher risk of breast cancer was reported initially by Xie and his colleagues, However, it still remains controversial. 22The Ile655Val (rs1136201) polymorphism of HER2 gene has been associated with breast cancer by many authors, 23 while some other studies did not show its correlation with the development of breast cancer risk in women. 24nce the overall results are inconsistent and controversial.
Due to controversies on HER1 and HER2 polymorphism association with breast cancer risk in various ethnicities, we aimed to conduct this study.We hypothesized that there could be an association of HER1-rs11543848 and HER2-rs1136201 with the risk of breast cancer in Khyber Pakhtunkhwa population.Therefore, the main aim of this study was to confirm the presence and association of HER1 and HER2 polymorphism with the risk of breast cancer.

| DNA extraction, SNP genotyping, and gel electrophoresis
The salting-out extraction method was used for the isolation of genomic DNA already adopted in our lab. 25Briefly, red blood cells lysis was performed by lysis buffer (TKM1) and Triton-X followed by incubation and centrifugation.The white blood cells were obtained in the form of pallet and nuclei lysis was done by TKM2 and Sodium dodecyl sulfate (SDS).After the incubation 6 M NaCl was added for the precipitation of protein.The Ethanol was used for the removal of excess salt and isopropanol was used for the precipitation of DNA.
The extracted DNA was air-dried and TE buffer was added for storage at −20°C until further use.The purity and concentration of DNA were determined by Nano drop (Thermo Fisher Scientific).
Amplification Refractory Mutation System PCR (ARMS-PCR) was used for SNPs genotyping, used at our lab. 25,26The specific outer and inner primer sequences (forward and reverse) were designed using online Primer blast software, to confirm homozygous and heterozygous alleles (Table 1).PCR mixture of 10 µL was prepared, including 5 µL master mix (5X) (Dream Taq, Thermo Fisher Scientific), 0.5 µL of each forward and reverse primers (1 µM of each primer), 3 µL ddH 2 O, and 1 µL of template DNA (50-100 ng).The amplified PCR products were confirmed with Gel electrophoresis using 1 KB DNA ladder on 2% agarose gel.

| Statistical analysis
The collected data were statistically analyzed using software such as   3).T A B L E 2 Allelic and genotypic frequencies of HER1-rs11543848 and HER2-rs1136201polymorphism in breast cancer patients and healthy control.
Genotype Breast cancer is the leading cancer in women worldwide.Many risk factors have been associated with the risk of breast cancer but recently due to the advent in molecular techniques, genetic predisposition of breast cancer have been extensively studied.Large number of studies have been conducted on HER1 and HER2 genes polymorphism and the risk of breast cancer on different populations.However, this research has revealed a landscape of conflicting and inconclusive findings, adding complexity to our understanding of these genetic factors' direct impact on breast cancer susceptibility. 10,24,27re in this study, we studied the association of HER1-rs11543848 and HER2-rs1136201 polymorphism with the risk of breast cancer in our population.A total of 314 women, containing 164 breast cancer patients and 150 age and gendermatched healthy controls were analyzed for SNPs genotyping using Tetra-ARMS-PCR.Interestingly, our results indicated that both HER1-rs11543848 and HER2-rs1136201 risk alleles and their heterozygous genotypes are highly significant (p < 0.05) with breast cancer risk (Table 2).
We further investigated the association between clinicopathological characteristics of breast cancer patients and these polymorphisms.Our analysis revealed that HER1-rs11543848 showed heightened but statistically nonsignificant associations with  3.
Previously, a study was conducted in the Egyptian population which revealed that HER1-rs11543848 and HER2-rs1136201 are significantly associated with the increased risk of breast cancer, similar to our results.However, their results diverged in terms of clinicopathological parameters, where no association was found.This disparity might stem from differences in population demographics, genetic variations, or environmental factors, which could influence how these genetic variations manifest in breast cancer development. 10Contradictory findings were observed in comparison to a study on Tunisian patients 28 , which reported a nonsignificant association between HER1-rs11543848 polymorphism and breast cancer.Conversely, they identified an association with tumor grade and nodal status.Such discrepancies could arise due to variations in sample sizes, ethnic differences, or specific genetic makeup within these distinct populations, contributing to contrasting observations regarding the polymorphism's role in breast cancer development and progression. 28Similarly, another case-control study highlighted an association between AG + GG genotypes of HER2-rs1136201 and higher breast cancer risk in patients younger than 45 years old.
However, our study presented an elevated association between genotypes of HER2-rs1136201 and patients over 45 years old.This disparity in age-specific associations could be influenced by diverse genetic backgrounds or lifestyle factors prevalent in these age groups, emphasizing the complexity of genetic factors' impact on breast cancer susceptibility across different age brackets.
Regarding the potential explanation for the association of HER2-rs1136201 polymorphism with breast cancer in our study, a hypothesis posits functional modifications rather than mere overexpression of HER2 protein.This suggests an increase in heterodimer formation, which could alter tyrosine kinase activity.
This theory underscores the need for further mechanistic studies to elucidate how these genetic variations lead to functional changes, impacting breast cancer susceptibility and disease progression.
It is important to note the inherent variability in somatic changes related to these polymorphisms across diverse ethnic groups, influencing their roles in breast cancer.This divergence underscores the complexity of genetic factors and necessitates cautious interpretation when comparing studies conducted in distinct ethnic populations, as genetic variations and their associations with breast cancer can vary significantly.
In essence, the differing outcomes in these studies emphasize the intricate interplay between genetic variations, environmental factors, and ethnic diversity in shaping the role of HER1-rs11543848 and HER2-rs1136201 polymorphisms in breast cancer susceptibility and clinicopathological characteristics.These complexities warrant further comprehensive research to unravel the precise mechanisms underlying these associations and their implications across diverse populations.

2 | MATERIALS AND METHODS 2 . 1 |
Patients' enrollment and ethical approval This case-control study was conducted during 2021-2022, containing 164 breast cancer patients and 150 age and gender-matched healthy controls.All the participants were enrolled from the Institute of Radiology and Nuclear Medicine (IRNUM) Hospital Peshawar, Pakistan, who had only breast cancer with no other complications.

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SOMBAL ET AL.Ethical approval was taken from the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar, Pakistan, and IRNUM hospital (IBGE, UAP/2022/002).A written informed consent was taken from all the participants after explaining the aim of the study.About 3 mL venous blood samples were taken in ethylenediaminetetraacetic acid tubes along with clinicopathological data.The blood was stored at −20°C for DNA extraction.
characteristics such as age group >45 years (OR = 1.6625, p = 0.2556), positive family history (OR = 7.400, p = 0.159), metastasis (p = 0.636), and luminal B subtype (OR = 8.336).In contrast, HER2-rs1136201 displayed significant associations with family history (p = 0.0001), metastasis (p = 0.001), luminal B subtype (OR = 14.82 p = 0.05), age group >45 years (OR = 2.285, p = 0.030), and TNBC patients (OR = 3.5556 p = 0.001), as outlined in Table morphism exhibited significant association with breast cancer risk in the Pashtun population of Khyber Pakhtunkhwa, Pakistan.These findings hold promise for advancing personalized medicine by potentially enabling more targeted screening and intervention strategies for individuals within this specific demographic.Moreover, they underscore the need for continued research in exploring these genetic variations' broader implications across different populations, paving the way for tailored preventive measures and more effective therapeutic interventions in the realm of breast cancer management.

Table 2
Statistical Package for Social Sciences (SPSS) version 16 to find the association of HER1 and HER2 genes polymorphisms with breast cancer risk.The clinicopathological, demographic characteristics, and genetic variants of breast cancer cases were detected by χ 2 test.The p < 0.05 was considered significant.Medcalc odd ratios calculator and 95% confidence intervals were used to figure out the possible association of HER1 and HER2 polymorphisms.Specific primers sequences designed for the detection of SNP genotype.